Protein synthesis is required for persistent forms of synaptic plasticity, including long-term potentiation (LTP). A key regulator of
LTP-related protein synthesis is mammalian target of rapamycin (mTOR), which is thought to modulate translational capacity by
also was shown to mediate plasticity-related translation, an effect that may involve regulation of the mTOR pathway. We studied the
interaction between the mTOR and ERK pathways in hippocampal LTP induced at CA3–CA1 synapses by high-frequency synaptic
of mTOR, increased in area CA1 stratum radiatum. This upregulation was detected in pyramidal cell dendrites and was blocked by
PI3K and ERK pathways. The role of ERK in regulating PDK1 and Akt, with their extensive effects on cellular function, has important
Key words: mammalian target of rapamycin; mitogen-activated protein kinase; dendrites; ERK; hippocampus; LTP; protein synthesis;
memory formation, and understanding the signaling network
how they can be disrupted. At least two phases of LTP can be
distinguished: a decremental phase (early LTP) that reflects only
posttranslational processes, and a stable phase [late LTP (L-
LTP)] that requires de novo protein synthesis (Frey et al., 1988;
Huang and Kandel, 1994; Osten et al., 1996; Tsokas et al., 2005).
The effect of synthesis inhibitors on LTP can be detected as soon
as 15–20 min after induction, suggesting that plasticity-related
proteins (PRPs) are produced rapidly and locally after stimula-
tion, and the dendritic expression of some proteins is increased
within 5 min after LTP induction (Ouyang et al., 1999; Tsokas et
al., 2005). Furthermore, dendrites severed from their cell bodies
are competent to express L-LTP (Kang and Schuman, 1996;
Cracco et al., 2005; Tsokas et al., 2005; Vickers et al., 2005) (but
see Frey et al., 1989), indicating that the local translation of den-
dritic mRNAs can supply the necessary PRPs.
LTP-related translation is controlled by coordinated mecha-
nisms that regulate mRNA availability and the activity of trans-
2006). Several studies have pointed to a key role for the protein
kinase mammalian target of rapamycin (mTOR), a regulator of
cell growth that facilitates translation of the terminal oligopyri-
midine (TOP) class of mRNAs (Meyuhas and Hornstein, 2000;
Tang et al., 2002; Cammalleri et al., 2003; Tsokas et al., 2005).
including ribosomal proteins and elongation factors, and the
mTOR-mediated synthesis of these components is thought to
try, Box 1215, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029. E-mail:
TheJournalofNeuroscience,May30,2007 • 27(22):5885–5894 • 5885
increase translational capacity, allowing cells to accommodate
periods of increased demand for new proteins (Meyuhas and
Hornstein, 2000). mTOR is present in the dendrites of hip-
pocampal neurons, and the dendritic expression of the TOP
mRNA-encoded elongation factor 1A (eEF1A) rapidly increases
in an mTOR-dependent manner after LTP induction; moreover,
L-LTP requires a period of mTOR activity (Tang et al., 2002;
with the hypothesis that L-LTP-inducing stimulation generates
dendritic translational capacity; however, eEF1A is the only TOP
mRNA product known to accumulate in neurons undergoing
The canonical pathway for mTOR activation begins with
phosphatidylinositide 3-kinase (PI3K), which generates phos-
phoinositides that recruit and colocalize phosphoinositide-
evidence that extracellularly regulated kinase (ERK) also plays a
role in the synaptically induced activation of mTOR is of partic-
ular interest, because ERK has been implicated in hippocampal
Atkins et al., 1998; Kelleher et al., 2004).
PI3K–mTOR pathway in LTP and its consequences for the ex-
pression of multiple TOP mRNA-encoded proteins. Our results
support the hypothesis that LTP-inducing stimulation increases
dendritic translational capacity by generating new synthetic ma-
and ERK at the level of PDK1.
Electrophysiology. Each male Sprague Dawley rat (6–8 weeks of age) was
deeply anesthetized with halothane and decapitated. The brain was rap-
following (in mM): 118 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25
NaH2PO4, 24 NaHCO3, and 15 glucose, bubbled with 95% O2/5% CO2.
The hippocampus was then quickly dissected out, and 500-?m-thick
slices were maintained in an interface chamber (ACSF and humidified
removal for electrophysiological recording. Slices were transferred to a
?s) were delivered with a bipolar stainless steel electrode placed in stra-
tum radiatum of the CA3 region, and the field EPSP (fEPSP) was re-
corded in the stratum radiatum of the CA1 region with electrodes filled
with ACSF (Re, 2–4 M?). The fEPSP was monitored by delivering stim-
uli at 0.033 Hz, the signal was low-pass filtered at 3 kHz and digitized at
20 kHz, and waveforms were collected and analyzed using either an
Axobasic routine or pClamp 9 (Molecular Devices, Foster City, CA). All
slices used in the experiments had spike thresholds ?2.5 mV. LTP was
induced by two 1-s-long trains of 100 Hz stimulation [high-frequency
sufficient to evoke 75% of the spike threshold. In all experiments, HFS
chamber, when the basal EPSP had been stable for at least 20 min. Each
experimental slice was paired with a sham-stimulated control, which
at room temperature in submersion maintenance chambers containing
min before HFS and for the remainder of the recording (5–30 min after
HFS, as indicated).
Western immunoblotting. Slices were removed from the recording
in some experiments the isolated stratum radiatum of CA1, was excised
in a cold room (4°C) under a dissection microscope. Samples were then
transferred to cold microcentrifuge tubes and stored at ?80°C for no
more than 3 d before assaying. Each excised region was homogenized in
35–50 ?l of ice-cold lysis buffer [in mM (unless indicated otherwise): 25
Tris-HCl, pH 7.4, 150 NaCl, 6 MgCl2, 2 EDTA, 1.25% NP40, 0.125%
SDS, 0.625% Na deoxycholate, 4 p-nitrophenyl phosphate, 25 Na fluo-
acid (phosphatase inhibitor cocktail I and II, 2 and 1%, respectively;
Calbiochem, La Jolla, CA), 1 phenylmethylsulfonyl fluoride (PMSF), 20
?g/ml leupeptin, and 4 ?g/ml aprotinin]. Protein determination was
performed using Bio-Rad (Hercules, CA) RC-DC Protein Assay kit. Ap-
bad, CA) and ?-mercaptoethanol were added to the homogenates, and
samples were boiled for 5 min. Samples were loaded on 8–10% SDS-
PAGE gel and resolved by standard electrophoresis. The gels were then
transferred onto nitrocellulose membranes (0.2 ?m pore size) at 4°C.
The membranes were blocked for at least 30 min at room temperature
with blocking buffer (BB) [5% nonfat dry milk in TBS containing 0.1%
Tween 20 (TBS-T)] and then probed overnight at 4°C using primary
follows (all were obtained from Cell Signaling Technology, Beverly, MA,
kinase (RSK), phospho(S241)-PDK1, phospho(T308)-Akt (Upstate Bio-
After washing in TBS-T (three washes, 5 min each), the membranes
were incubated with horseradish peroxidase-conjugated anti-rabbit or
anti-mouse IgG (1:5000; Pierce Biotechnology, Rockford, IL), and pro-
teins were visualized by chemiluminescence (ECL Western Blotting
Analysis System; GE Healthcare, Arlington Heights, IL). Densitometric
analysis of the bands was performed using NIH Image, and values were
normalized to actin. t tests were used to compare samples from experi-
mental slices to their respective controls, and summary data are pre-
sented as group means with SE bars.
Immunohistochemistry and confocal microscopy. Immediately after re-
cording, 500 ?m slices were placed in ice-cold 4% paraformaldehyde/
vibratome. Free-floating sections were blocked with 10% normal goat
serum, 1% BSA, and 0.1% Na azide in PBS. The sections then were
incubated overnight at 4°C with primary antibody in 1% BSA (in some
experiments, two antibodies from different species were incubated to-
gether; otherwise, serial immunohistochemistry was performed). Anti-
bodies used for immunohistochemistry included: phospho(Thr421/
Ser424)-p70S6K (Santa Cruz Biotechnology, Santa Cruz, CA),
phospho(Thr389)-p70S6K (Cell Signaling Technology, Beverly, MA),
eEF1A (Upstate Biotechnology), rpS6 (Cell Signaling Technology),
PABP (gift from J. Brosius, University of Mu ¨nster, Mu ¨nster, Germany),
and ?-tubulin (Sigma-Aldrich).
Fixed tissue sections were probed with primary antibodies, including
?-tubulin to visualize dendrites. After washing in PBS, sections were
incubated in 1% BSA with secondary antibodies complexed to either
Alexa Fluor 568 or Alexa Fluor 488 (both used at 1:250; Invitrogen).
Where multiple monoclonal primary antibodies were used, the slices
sequentially incubated with the next primary antibody. After extensive
510 confocal microscope at a scanning depth of 1.9 ?m (200?) or 0.9
?m (400?). Alexa Fluor 488 immunofluorescence was detected with an
was used with a 560–615 nm long-pass emission filter to visualize Alexa
Fluor 568. All parameters (pinhole, contrast, and brightness) were held
constant for all sections from the same experiment. For double-label
experiments, scans at different wavelengths were digitally merged. Mul-
tiple overlapping fields within the same focal plane were imaged and
assembled to construct composites of the CA1 region.
5886 • J.Neurosci.,May30,2007 • 27(22):5885–5894Tsokasetal.•MAPKRegulationofthemTORPathwayinLTP
state Biotechnology) was added to the supernatant and incubated for at
with samples for at least 3 h at 4°C. Immunocomplexes were then spun
down and washed five times with ice-cold lysis buffer. Kinase reactions
were started by adding 0.9 ?g of recombinant human 4EBP1 (gift from
MgCl2, 1 Na fluoride, 2 Na pyrophosphate, 10 ?-glycerophosphate,
phosphatase inhibitor cocktail I and II (2 and 1%, respectively; Calbio-
2 ATP. The tubes were then placed in a 30°C water bath and vortexed
every few minutes, and the reaction was stopped after 20 min by adding
4EBP (Cell Signaling Technology).
To evaluate whether the hypothesis that increased translational
of L-LTP causes a general increase in the expression of TOP
mRNA products, as established previously for eEF1A (Tsokas et
al., 2005). The proteins that we studied were eEF2, rpS6, and
PABP, all of which have been implicated in the response of neu-
rons to plasticity-inducing stimulation (Marin et al., 1997; Wu
and Cline, 1998; Scheetz et al., 2000; Huang et al., 2002; Kelleher
et al., 2004; Takei et al., 2004; Atkins et al., 2005; Alarcon et al.,
2006). Within 30 min after late L-LTP-inducing HFS, immuno-
reactivity for all of these proteins increased twofold to threefold
in homogenates of excised area CA1 (Fig. 1A). To confirm that
these changes required de novo synthesis and were mediated by
mTOR, as previously shown for eEF1A, we delivered HFS in the
presence of the translation inhibitor anisomycin or the mTOR
inhibitor rapamycin. Under these conditions, HFS failed to in-
crease the expression of eEF2, rpS6, or PABP (Fig. 1A). In con-
mycin or rapamycin (Fig. 1B), indicating that the inhibitors
prevented the de novo translation of the TOP mRNAs. Thus, the
induction of L-LTP resulted in a coordinate and mTOR-
mediated synthesis of translational machinery, consistent with
the proposed role of mTOR in regulating translational capacity.
We and others have shown that translation-dependent LTP can
be detected within 20 min after HFS (Osten et al., 1996; Kelleher
et al., 2004; Tsokas et al., 2005). If the synthesis of LTP-related
proteins depends on an mTOR-mediated boost in translational
capacity, then increases in the expression of TOP mRNA-
encoded proteins should generally be apparent soon after stimu-
lation. We previously reported that eEF1A is upregulated within
5 min after HFS, indicating that increased translational capacity
could comprise an early event in the process leading to the syn-
pathway, as shown by their sensitivity to anisomycin (Aniso; 10 ?M) and rapamycin (Rapa; 1 ?M), respectively. Immunoreactivity, here and in subsequent figures, is expressed relative to
than in controls (left panels), and these effects were blocked by rapamycin (right panels). s.o., s.p., and s.r. indicate strata oriens, pyramidale, and radiatum, respectively. The images are
Tsokasetal.•MAPKRegulationofthemTORPathwayinLTPJ.Neurosci.,May30,2007 • 27(22):5885–5894 • 5887
whether the expression of eEF2, rpS6, and PABP also increases
rapidly in stimulated CA1 dendrites, we first performed immu-
noblots on homogenates of stratum radiatum that had been mi-
crodissected from slices frozen 5 min after strong HFS. This ex-
cision was conservative, sacrificing some proximal stratum
dal cell bodies. Homogenates from stimulated slices showed in-
creases in eEF2, rpS6, and PABP immunoreactivity that were
blots of complete CA1 regions (Fig. 1, compare C and A). The
the increase at 30 min in CA1; for rpS6, 9.1-fold higher; and for
PABP, 3.5-fold higher. The stronger upregulation in stratum ra-
diatum suggests that the major component of the increase in
protein expression was contributed by the apical dendrites of
CA1 neurons. Moreover, the short latency of the increases in
increase in translational capacity contributes to L-LTP.
To further investigate the cellular location of TOP mRNA
sion of rpS6 and PABP after strong HFS, using immunohisto-
later, immunoreactivity for both TOP mRNA-encoded proteins
was intensified in well-defined dendrites of stratum radiatum
relative to slices that had received control stimulation (Fig. 1D).
throughout the proximal-distal extent of the apical dendrites.
HFS also increased the expression of both proteins in the cell
bodies, where there was substantial immunoreactivity even in
control slices, and diffusely in the dendrites of stratum oriens.
Thus, the HFS-induced expression of rpS6 and PABP showed a
somatodendritic distribution at 5 min that was similar to that
et al., 2005) (Figs. 1D, bottom, 3B,C). Considering the sparse
innervation of the pyramidal cell bodies by excitatory synapses
(Megias et al., 2001), the increased somatic expression of TOP
mRNA-encoded proteins likely reflects retrograde signaling me-
an upstream signaling component.
To confirm that the HFS-induced dendritic expression of
rpS6 and PABP expression depended on activity in the mTOR
pathway, we challenged these effects with rapamycin. In agree-
ment with our immunoblot data, rapamycin blocked the soma-
todendritic increases in rpS6 and PABP immunoreactivity after
strong HFS (Fig. 1D, right panels). Thus, the mTOR pathway
mediates a rapid and general increase in the dendritic expression
of TOP mRNA-encoded components of the translational ma-
dependent component of LTP emerges within 20 min after HFS
(Osten et al., 1996; Tsokas et al., 2005).
In addition to being a TOP mRNA product, rpS6 is subject to
phosphorylation by the mTOR effector p70S6K. Kelleher et al.
(2004) found that HFS increased rpS6 phosphorylation at an
mTOR-dependent site, and that this effect required an intact
in LTP. We therefore tested whether inhibitors of the ERK path-
TOP mRNA products eEF1A, eEF2, rpS6, and PABP. Immuno-
blots of CA1 homogenates showed that the ERK kinase (MEK)
inhibitors PD98059 and U0126 prevented HFS-induced changes
in the expression of all four proteins (Fig. 2A). This dependence
proteins, the basal levels of which were unaffected by ERK inhi-
bition (Fig. S1, available at www.jneurosci.org as supplemental
material). We also tested the ability of ERK inhibitors to prevent
L-LTP under our conditions and found that synaptic potentia-
tion returned to baseline within 2 h after HFS delivered in the
studies showing that ERK activity is required for LTP induction
for the ERK-dependent component of LTP is similar to that seen
cin, using this HFS protocol or a similar one (Osten et al., 1996;
Tsokas et al., 2005). In agreement with Kelleher et al. (2004),
sis by regulating the mTOR pathway.
To establish that ERK does in fact mediate the activation of
mTOR in LTP, and to elucidate the nature of the interaction
whether ERK mediates the HFS-induced phosphorylation of
al., 1997) (but see Stolovich et al., 2002). There are at least eight
phosphorylatable sites in p70S6K, and the activation of this en-
tiple protein kinases, including mTOR (Pullen and Thomas,
1997). Among the mTOR-dependent sites is T389 in the hydro-
indicate p70S6K activation, and we have shown previously that
HFS increases phospho-T389 immunoreactivity in CA1 den-
requires the previous phosphorylation of T421/S424 in the auto-
inhibitory region of p70S6K, a site shown to be ERK-dependent
in some studies (Zhang et al., 2001; Lehman et al., 2003). To
determine whether ERK is required for the phosphorylation of
these sites in LTP, we performed immunoblots from CA1 ho-
mogenates using phospho-specific antibodies. In vehicle-treated
slices, immunoreactivity for both phospho-T389 and phospho-
T421/S424 increased after the delivery of HFS (Fig. 3A), whereas
total p70S6K expression was not affected (Fig. S2, available at
mycin pretreatment prevented the HFS-induced phosphory-
lation of T389 but left the phosphorylation at T421/S424 in-
tact. In contrast, HFS delivered in the presence of U0126 failed
to induce phosphorylation at either site. These findings agree
with those showing that phosphorylation of T421/S424 is ERK
dependent and indicate that the ERK and mTOR pathways
might interact at the level of p70S6K through an intramolec-
ular mechanism. However, these data do not rule out the pos-
sibility of a more upstream effect of ERK on the mTOR
To determine whether ERK mediates the HFS-induced stim-
ulation of the mTOR pathway in dendrites, slices were fixed for
immunohistochemistry 30 min after HFS. Pretreatment with
U0126 blocked the phosphorylation of dendritic p70S6K at both
T389 and T421/S424, as well as the increase in dendritic eEF1A
expression (Fig. 3B,C). These findings agree with our immuno-
blot data, as does the observation that pretreatment with rapa-
mycin prevented the effect on eEF1A without blocking phos-
5888 • J.Neurosci.,May30,2007 • 27(22):5885–5894Tsokasetal.•MAPKRegulationofthemTORPathwayinLTP
phorylation at T421/S424-p70S6K (Fig. 3C). Thus, ERK is
required for the activation of p70S6K in the dendrites after HFS.
As noted, the data in Figure 3 are consistent with an intramolec-
ular p70S6K mechanism, but they are also compatible with an
alternative hypothesis: that ERK controls mTOR at a more up-
stream component, independent of its role in the phosphoryla-
tion of p70S6K at T424/S424 (Fig. 4A) (Fro ¨din et al., 2000; Roux
et al., 2004; Ma et al., 2005). mTOR activity often correlates with
its phosphorylation at S2448, which is increased in cells that ex-
press constitutively active Akt (Nave et al., 1999; Sekulic et al.,
2000) (but see Holz and Blenis, 2005). To examine the possible
entry of ERK upstream of mTOR, we tested whether the phos-
phorylation of S2448-mTOR in response to HFS requires ERK
activity. As shown in Figure 4B, the pretreatment of slices with
U0126 prevented HFS-induced mTOR phosphorylation in area
CA1 (note that total mTOR expression was unaffected by HFS)
(Fig. S2, available at www.jneurosci.org as supplemental mate-
rial). To confirm the conclusion that ERK regulates mTOR sub-
sequent to the induction of late LTP, we assayed the activity of
mTOR immunoprecipitated from CA1 regions, using recombi-
a substrate. The ability of mTOR to phosphorylate 4EBP1 was
increase, in agreement with our mTOR phosphorylation results
and showing that the activation of mTOR by HFS is ERK
In the canonical pathway for mTOR regulation, PI3K-generated
phosphoinositides colocalize PDK1 and its substrate, Akt, at the
cell membrane. Phosphorylated Akt, in turn, indirectly activates
TSC2, also known as tuberin (Inoki et al., 2002; Manning et al.,
2002; Potter et al., 2002). This pathway is functional in the hip-
tor (mGluR) stimulation (Hou and Klann, 2004). Furthermore,
PI3K activity is necessary for synaptically induced LTP and for
al., 2002; Opazo et al., 2003; Hou and Klann, 2004).
The mechanism by which ERK regulates mTOR in LTP is
unknown, but studies in other systems have identified two pro-
teins in the PI3K-mTOR pathway where ERK might enter. First,
ERK can indirectly phosphorylate and activate PDK1, an effect
that is mediated by the ERK substrate RSK (also known as
p90S6K) (Fro ¨din et al., 2000). At a more downstream site, ERK
can phosphorylate and inactivate TSC2, either directly or
ularly interested in the possibility that PDK1 activity might be
regulated by ERK, because PDK1 has extensive effects on signal
transduction and gene expression (Vanhaesebroeck and Alessi,
2000). PDK1 is stimulated upon phosphorylation by RSK, and
this effect requires that RSK be activated by ERK (Fro ¨din et al.,
2000). If this mechanism is important in late LTP, then PDK1
activity should be increased by HFS in an ERK-dependent man-
the ability of HFS to induce PDK1 autophosphorylation at S241
(Wick et al., 2003). In immunoblots of CA1 homogenates, im-
munoreactivity for S241-PDK1 was found to increase after HFS
mTOR activity was blocked (Tsokas et al., 2005), whereas LTP was stable for 2 h in vehicle-
treated controls (Veh) (n ? 5). The traces show superimposed fEPSPs recorded during the
baseline period and 2 h after HFS (arrows) in a vehicle-treated control slice (left) and a slice
ERK activity is required for the upregulation of translational proteins in LTP. A,
Tsokasetal.•MAPKRegulationofthemTORPathwayinLTP J.Neurosci.,May30,2007 • 27(22):5885–5894 • 5889
(but not the expression of total PDK1) (Fig. S2, available at
www.jneurosci.org as supplemental material), and this increase
was blocked when HFS was delivered in the presence of ERK
tion of Akt at T308, which is obligatory for the activation of Akt
and directly mediated by PDK1 (Alessi et al., 1997a; Chan et al.,
1999). In response to HFS, immunoreactivity to T308-Akt in-
creased more than twofold and in an ERK-dependent manner
(Fig. 5B). Finally, RSK phosphorylation was probed at the ERK-
dependent site S386. As shown in Figure 5C, HFS caused a rapid
hyperphosphorylation at S386-RSK, and this effect was blocked
by U0126 and PD98059, whereas total RSK expression was not
affected by HFS (Fig. S2, available at www.jneurosci.org as sup-
plemental material). In control experiments with unstimulated
slices, inhibition of ERK did not affect the basal phosphorylation
of RSK, Akt, or PDK1 at these sites (Fig. S1, available at www.
jneurosci.org as supplemental material), indicating that HFS-
induced phosphorylation of these proteins was specifically
blocked by ERK inhibitors under our conditions. Together,
these data support the hypothesis that ERK regulates mTOR
through the RSK-dependent activation of PDK1 and Akt.
Akt, mediated by the association of their pleckstrin homology
domains with PI3K-generated phosphoinositides (Currie et al.,
1999). This requirement is consistent with the ability of PI3K
2003), and we found that LTP induced by our protocol was sim-
ilarly PI3K dependent (Fig. S3, available at www.jneurosci.org as
et al. (2003) also observed that PI3K mediated the HFS-induced
increase in ERK activity. Coupled with our data, such an effect
PI3K-mTOR pathways in LTP. To examine this issue, we mea-
sured the PI3K-dependent component of ERK pathway activity
but not to rapamycin (Rapa; 1 ?M), whereas both inhibitors prevented the HFS-induced rise in eEF1A expression (bottom two images). These images are representative of three independent
HFS-induced phosphorylation of the mTOR substrate p70S6K is ERK dependent. A, In CA1 regions from slices frozen 30 min after HFS, p70S6K was hyperphosphorylated at the
inhibitory domain, along with the known obligatory phosphorylation sequence. Phos-
phorylation at T389 is rapamycin-sensitive, whereas ERK mediates phosphorylation at
T421/S424, indicating that ERK might control the mTOR-related activation of p70S6K
phosphorylation of T389 is that mTOR activity depends on ERK (dashed line). B, mTOR
phosphorylation by HFS requires ERK activity. Immunoblots for phospho(S2448)-mTOR
were performed on lysates of CA1 from slices frozen 30 min after stimulation. The HFS-
induced phosphorylation at S2448 was blocked in slices treated with 20 ?M U0126. The
activity of mTOR purified by immunoprecipitation from CA1 areas was assayed by phos-
pletely blocked by the MEK inhibitors, and rapamycin reduced mTOR activity below
HFS activates mTOR in an ERK-dependent manner. A, The ERK and mTOR
5890 • J.Neurosci.,May30,2007 • 27(22):5885–5894Tsokasetal.•MAPKRegulationofthemTORPathwayinLTP
expected, the PI3K inhibitor LY294002 (20 ?M) reduced the
HFS-induced increase in phospho-MEK and phospho-ERK.
However, the phosphorylation state of MEK and ERK in un-
significantly changed after stimulation. These findings contrast
phospho-ERK in unstimulated mouse hippocampus (Opazo et
al., 2003). The basis for this difference is not known, but one
possibility is that basal PI3K activity might be higher under our
conditions, perhaps reflecting a species-related variation. Sub-
stantial PI3K activity in unstimulated slices is likely to support
some background colocalization of PDK1 and Akt, which could
allow signals in the ERK pathway to propagate to mTOR (see
Discussion). It is noteworthy that the twofold increase in ERK
that reported by Opazo et al. (2003), suggesting that the contri-
bution of PI3K to basal ERK activity does
not reduce the dynamic range of the ERK
pathway that is available for stimulation
on the regulation of the PI3K-mTOR
pathway by ERK, indicate that reciprocal
interactions between the PI3K and ERK
translation at the level of PDK1 (Fig. 5E).
tional machinery is observed in many cell
types as an early response to stimuli that
induce cell growth, and this process de-
control over the expression of TOP
mRNA-encoded proteins (Meyuhas and
demand production of translational ma-
chinery would be to reduce the metabolic
cost of maintaining excess capacity under
conditions where relatively little protein
synthesis is needed and to add capacity at
specific places in preparation for a period
of increased demand. In the case of pro-
tein synthesis in the dendrites of a CA1
pyramidal cell, with a total dendritic
length of ?13,500 ?m (Ishizuka et al.,
1995), the advantages of regulating trans-
lational capacity are apparent.
Among the TOP mRNA-encoded pro-
teins studied here, PABP is interesting
from the standpoint of LTP. PABP binds
to the poly-A tail of mRNA and conse-
quently acts at the initiation step to in-
lengthen poly-A tails (Wells et al., 2001),
and under such conditions PABP expres-
sion might become rate-limiting for poly-
A-dependent translation. Thus, by synthesizing PABP, the neu-
ron could match the supply of this protein to the demands
imposed by the dynamics of polyadenylation.
The ability of HFS to increase eEF1A and eEF2 suggests that
the process of elongation also is upregulated in LTP. Protein
synthesis is commonly regulated at the initiation step; however,
in an environment where basal translational capacity is low, it is
The dendrite is apparently such a low-capacity environment,
shaft and spines (?1 per synapse) (Sutton and Schuman, 2006).
However, the control of elongation in synaptic plasticity is com-
plex, because some forms of stimulation result in the inhibition
of eEF2 (Scheetz et al., 2000).
encoded proteins occurs throughout the apical dendrites, sug-
gesting that translational capacity is boosted at a distance from
a component of MEK/ERK phosphorylation. Incubation with the PI3K inhibitor LY294002 (LY; 20 ?M) reduced both basal and
black are coregulated by the PI3K and ERK pathways (shown in blue and green, respectively). The arrows do not necessarily
tase (Peterson et al., 1999)], and some known interactions have been excluded for the sake of clarity (e.g., mTOR facilitates
Tsokasetal.•MAPKRegulationofthemTORPathwayinLTP J.Neurosci.,May30,2007 • 27(22):5885–5894 • 5891
the activated synapses. How could a widespread increase in den-
dritic translational capacity contribute to synapse-specific in-
creases in LTP-related proteins? One possibility is that the in-
which can be used only at synapses that have been “tagged” by
appropriate stimulation (Frey and Morris, 1997; Barco et al.,
2002; Govindarajan et al., 2006). An alternative hypothesis pos-
lationally repressed and become derepressed specifically at syn-
apses that have been appropriately stimulated (Krichevsky and
Kosik, 2001; Blitzer et al., 2005). In this way, a wide distribution
of synapses to synthesize PRPs rapidly, locally and on demand,
ensuring the input-specificity of synaptic plasticity. Experiments
that have addressed the issue of local protein synthesis in LTP
(Frey and Morris, 1997, 1998) and others suggesting that LTP
depends, at least in part, on local protein synthesis (Barco et al.,
2002; Alarcon et al., 2006). Additional studies are needed to re-
solve the roles of distributed and local protein synthesis in LTP,
and how these processes interact.
Multiple regulatory processes at the transcriptional, transla-
tional, and posttranslational levels are simultaneously active in
LTP, and a major challenge is to understand how these control
mechanisms are coordinated by upstream signaling molecules.
Among these key signaling components is ERK, which mediates
LTP-related protein synthesis and transcription and regulates
membrane excitability (Impey et al., 1998; Davis et al., 2000;
Yuan et al., 2002; Kelleher et al., 2004). Our finding that ERK
the PI3K–mTOR pathway is significant, because the HFS-
induced regulation of PDK1 helps to explain how ERK coordi-
nates the diverse cellular processes that underlie synaptic plastic-
ity. PDK1 has been described as a “master” regulator, because it
activates several protein kinases, including some that have been
implicated in synaptic plasticity, such as cyclic AMP-dependent
protein kinase and protein kinase C (Cheng et al., 1998; Chou et
al., 1998; Dutil et al., 1998; Mora et al., 2004). Moreover, the
PDK1-dependent Akt similarly regulates multiple translational
and transcriptional processes (Impey et al., 1998; Gingras et al.,
1999; Meyuhas and Hornstein, 2000; Brazil and Hemmings,
2001; Wang et al., 2001; Banko et al., 2004). The present investi-
gation is the first to demonstrate the importance of ERK in reg-
ulating the PDK1 and Akt pathways in LTP.
There has been some controversy regarding the regulation of
PDK1 activity and its role in controlling the mTOR pathway,
because previous studies showed the isolated protein to be con-
stitutively active (Alessi et al., 1997b). Both PDK1 and Akt con-
tain pleckstrin homology domains and therefore associate with
3?-phosphoinositides in the cell membrane, so it has been sug-
gested that PI3K regulates the mTOR pathway by bringing con-
stitutively active PDK1 together with Akt. However, studies in
intact cells have shown extracellular stimuli to increase PDK1
activity (Wick et al., 2000; Park et al., 2001; Sato et al., 2002),
suggesting the presence of regulatory proteins in vivo. Several
cellular mechanisms for increasing PDK1 activity have been de-
scribed, including the binding of PDK1 to a docking site in the
hydrophobic domain of RSK, which is generated upon RSK au-
tophosphorylation at S386 by its C-terminal kinase (CTK) do-
by ERK, which thus controls the interaction between PDK1 and
the hydrophobic domain of RSK, an association that should dra-
matically increase PDK1 activity (Biondi et al., 2000). The very
low basal activity of the major RSK isoform in the hippocampus,
along with its strong response to stimulation that activates ERK,
suggest that the RSK–PDK1 interaction is an important factor in
controls the mTOR pathway in LTP (Fig. 5).
We have shown that ERK is required for the HFS-induced
ERK is not necessary for mTOR activation under certain other
stimulation conditions. For example, the treatment of cultured
neurons with brain-derived neurotrophic factor increases Akt
phosphorylation even in the presence of PD98059 (Takei et al.,
2001; Schratt et al., 2004). These differences in the sensitivity of
mTOR to ERK inhibitors are interesting and might reflect the
PDK1 to the membrane, where it can phosphorylate Akt and
other membrane-associated substrates, whereas the efficacy of
this process should be modulated by ERK-dependent changes in
the intrinsic activity of PDK1. Thus, any treatment that strongly
stimulates PI3K, and thereby causes extensive colocalization of
PDK1 and Akt, might allow PDK1 with low intrinsic activity to
substantially phosphorylate Akt, and ERK would be unnecessary
for the stimulation of the mTOR pathway. Such a cooperative
relationship between PI3K and ERK might explain the observa-
tion by Opazo et al. (2003) that PI3K inhibitors prevented HFS-
induced LTP in mouse hippocampus (Winder et al., 1999),
whereas MEK inhibitors were ineffective. Conversely, when
PDK1 is stimulated by ERK, even a modest recruitment of PDK1
might be enough to activate mTOR, and this type of regulation
would be ERK dependent. This model suggests the intriguing
possibility that stimulus-induced changes in ERK activity might
affect mTOR even when PI3K activity remains at its basal level,
can induce p70S6K phosphorylation without any increase in
PI3K activity (Ma et al., 2005). Furthermore, PI3K is subject to
regulation by ?? subunits and tyrosine kinase activity (Murga et
al., 1998; Patapoutian and Reichardt, 2001); thus, there could be
a dynamic relationship between PI3K and ERK in which neuro-
transmitters and growth factors set the level of PI3K activity, and
this in turn determines the ability of ERK signaling to propagate
to mTOR. The concept of ERK playing an executive role in reg-
ulating mTOR is clearly distinct from the canonical mechanism,
and it will be interesting to identify conditions where neuronal
mTOR is activated by stimuli that specifically stimulate ERK.
of PDK1 and its substrate Akt on gene expression, the present
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