MOLECULAR AND CELLULAR BIOLOGY, Oct. 2011, p. 4063–4075
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 19
Spatiotemporally Regulated Protein Kinase A Activity Is a Critical
Regulator of Growth Factor-Stimulated Extracellular
Signal-Regulated Kinase Signaling in PC12 Cells?
Katie J. Herbst,1Michael D. Allen,1† and Jin Zhang1,2,3*
Department of Pharmacology and Molecular Sciences,1Solomon H. Snyder Department of Neuroscience,2and Department of
Oncology,3The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received 7 April 2011/Returned for modification 2 May 2011/Accepted 19 July 2011
PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal
growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell prolifer-
ation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation.
While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained
ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein
kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized
to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma
membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail
to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining
this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotem-
poral regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity
in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA
activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the
growth factor signaling specificity achieved by NGF and EGF in PC12 cells.
To ensure proper conversion of a specific environmental
input into a distinct cellular output, cells exploit a number of
molecular mechanisms to tightly regulate signal transduction
in space and time. In PC12 cells, specific controls of the dura-
tion of the activity of extracellular signal-regulated kinase
(ERK), a canonical mitogen-activated protein kinase (MAPK),
are believed to help determine distinct cell fates (32). Specif-
ically, activation of epidermal growth factor receptor (EGFR)
by epidermal growth factor (EGF) leads to transient ERK
activity and cell proliferation, whereas nerve growth factor
(NGF) binding to and activating its receptor, TrkA, leads to
sustained ERK activity and signals the cells to differentiate
(23). An accepted model for the growth factor (GF) signaling
specificity in these cells involves the activation of specific
GTPases capable of activating the Raf family of kinases, which
activate MEK, the upstream activator of ERK. In particular,
while both EGF and NGF can transiently activate the GTPase
Ras to recruit Raf to the plasma membrane, where it can be
activated, only NGF activates Rap1, a cyclic AMP (cAMP)-
regulated GTPase also capable of activating Raf (23). Since
this NGF-induced Rap1 activation is sustained, it is suggested
that the selective activation of Rap1 by NGF but not EGF
leads to the sustained phase of ERK activity and the initiation
of neurite outgrowth. Furthermore, EGF-stimulated ERK
negatively regulates Raf activity, whereas NGF-stimulated
ERK exerts positive feedback on Raf activity, further contrib-
uting to the transient and sustained duration of ERK activity as
a result of the respective stimuli (11, 22).
As an added level of complexity in signal transduction reg-
ulation, many canonical signaling cascades are subject to cross
talk, in which the molecular players of one pathway alter the
state of another. For example, it is known that the ERK path-
way and the cAMP-mediated signaling pathway are intricately
connected (28). While the precise regulation that these path-
ways have on one another is complex and cell type specific (28),
it is widely accepted that in PC12 cells, two cAMP effectors,
namely, cAMP-dependent protein kinase (PKA) and exchange
protein directly activated by cAMP (Epac), can indirectly ac-
tivate the Raf/MEK/ERK cascade (4, 5, 28).
Intracellular cAMP is enzymatically produced from ATP by
adenylyl cyclases, either transmembrane adenylyl cyclase
(tmAC) or soluble adenylyl cyclase (sAC) (10), and degraded
by phosphodiesterases (PDEs), of which there are 11 known
isoforms (2). In PC12 cells, NGF binds to TrkA, which acti-
vates sAC to produce cAMP (26). Subsequently, activated
PKA and Epac converge to activate Rap1 (26), the aforemen-
tioned mediator of sustained ERK activity (36, 37). In contrast,
EGF was not known to increase cAMP or activate PKA in
PC12 cells (14). Interestingly, a number of studies have shown
that when EGF is used in conjunction with cAMP-elevating
agents, neurite outgrowth can be induced in PC12 cells (9, 14,
15, 35). These studies suggest that cAMP-mediated signaling
may play a role in GF signaling specificity in PC12 cells and
point to a simple model showing that lack of cAMP-mediated
cross-regulation specifies the transient ERK activity stimulated
by EGF. However, GF-stimulated cAMP increases and PKA
* Corresponding author. Mailing address: 725 N. Wolfe Street, Hun-
terian 307, Baltimore, MD 21205. Phone: (410) 502-0173. Fax: (410)
955-3023. E-mail: firstname.lastname@example.org.
† Present address: Clinical Endocrinology Branch, National Insti-
tute of Diabetes and Digestive and Kidney Diseases, National Insti-
tutes of Health, Bethesda, MD 20892.
?Published ahead of print on 1 August 2011.
activities have not been characterized in this system, and this
model remains to be tested.
Characterization of cAMP-mediated signaling in a number
of cell systems has revealed complex spatiotemporal regulation
that is often achieved by the formation of localized signaling
complexes containing the key regulatory and effector mole-
cules involved in cAMP signaling. One critical component of
these complexes, which are frequently formed with the help of
scaffolding proteins known as A-kinase anchoring proteins
(AKAPs), are PDEs (1). Localization of PDEs to distinct in-
tracellular locales results in the formation of discrete cAMP
gradients which are able to control the signaling of PKA and
Epac with high specificity (1). Proper compartmentalization of
PKA- and Epac-mediated signaling is necessary for normal
cellular function and regulates processes such as excitation and
contraction of cardiomyocytes and insulin secretion in pancre-
atic ? cells (13, 29). In hippocampal neurons, PDEs play an
important role in establishing localized pools of cyclic nucleo-
tides in undifferentiated neurites, which ultimately regulates
the process of axon and dendrite formation (24). Although
cAMP-mediated signaling contributes to specifying the cell
fates of PC12 cells as discussed above, the spatiotemporal
regulation of cAMP signaling and its functional roles in this
cell system have not been fully characterized.
In this study we investigated the spatiotemporal regulation
of GF-mediated PKA activity in PC12 cells by employing ge-
netically encoded fluorescence resonance energy transfer
(FRET)-based biosensors targeted to distinct subcellular lo-
cales. In doing so, we observed that both NGF and EGF
stimulate PKA activity at the plasma membrane with discrete
temporal activity patterns, but both stimuli fail to activate the
cytosolic pool of PKA. Using pharmacological perturbations,
we identify PDE3 to be a critical regulator of this GF-stimu-
lated compartmentalized PKA activity. Moreover, PKA and
PDE3 are shown to regulate both the onset and duration of
GF-stimulated ERK activity. When PDE3 is inhibited and the
spatiotemporal regulation of PKA is disrupted, PC12 cells
treated with EGF display heightened durations of nuclear
ERK activity. Together these findings reveal the distinct NGF-
and EGF-induced spatiotemporal patterns of PKA activity as a
novel level of regulation underlying the precise GF signaling
specificity in PC12 cells.
MATERIALS AND METHODS
Cellular culture and transfection. PC12 cells were grown in Dulbecco modi-
fied Eagle medium (DMEM) cell culture medium supplemented with 10% fetal
bovine serum (FBS) and 5% donor horse serum (DHS) at 37°C with 5% CO2.
Cells were plated onto 35-mm glass-bottom dishes for imaging or 15-cm culture
dishes for fractionation studies. When necessary, cells were transfected at 60 to
70% confluence via Lipofectamine 2000 (Invitrogen). Prior to treatment, cells
were grown in reduced serum medium (5% FBS, 1% DHS) for 24 h.
Cellular imaging and analysis. For imaging, cells were washed once and then
imaged in Hanks’ balanced salt solution on a Zeiss Axiovert 200 M microscope
with a cooled charge-coupled-device camera (MicroMAX BFT512; Roper Sci-
entific, Trenton, NJ) controlled by METAFLUOR software (Molecular Devices,
Sunnyvale, CA). Dual emission ratio imaging was performed using a 420DF20
excitation filter and a 450DRLP dichroic mirror and appropriate emission filters,
475DF40 for cyan fluorescent protein (CFP) and 535DF25 for yellow fluorescent
protein (YFP). Cells were treated with drug as indicated, and images were
analyzed using ImageJ software (NIH).
PC12 cell fractionation and Western blots. For PC12 cell fractionation, cells
were harvested in TNE buffer containing 2 mM each of phenylmethylsulfonyl
fluoride (PMSF), NaF, and NaVO4, 10 nM calyculin A, and a protease inhibitor
cocktail (Roche) and lysed in a Dounce homogenizer. Lysate was spun at 5,000 ?
g for 10 min, and the supernatant was then spun at 13,000 ? g for 35 min. The
supernatant (cytosolic fraction) was separated from the pellet (membrane frac-
tion), and the pellet was solubilized in TNE containing 1% Triton X-100. Bicin-
choninic acid (BCA) assay (Pierce) was used to detect the total protein concen-
tration in each fraction. Each fraction contained 40 ?g of total protein, was
separated on 7.5% SDS-PAGE, and then was transferred to nitrocellulose mem-
branes. For Western blots, 24 h prior to treatment, PC12 cells were switched into
reduced serum medium and then treated as indicated. Cells were then harvested
and lysed in radioimmunoprecipitation assay (RIPA) buffer containing 2 mM
each of PMSF, NaF, and NaVO4, 10 nM calyculin A, and a protease inhibitor
cocktail. Lysate was incubated on ice for 30 min and then spun at 13,000 ? g at
4°C for 30 min. The total protein concentration was detected by BCA assay.
Proteins were separated on 7.5% SDS-PAGE and then transferred to nitrocel-
lulose membranes. In both cases, membranes were blocked in 5% BSA and
incubated overnight with primary antibody. Membranes were washed and incu-
bated with secondary antibody, and ECL Western blotting substrate (Pierce) was
used for detection. Densitometric analysis was performed using ImageJ (NIH).
For the fractionation studies, the total PDE3 in each fraction was quantified by
summing the PDE3 band intensity from each fraction.
NGF and EGF induce distinct spatiotemporal patterns of
PKA activity. To begin our studies, we looked at the activation
of PKA downstream of TrkA and EGFR at the plasma mem-
branes of PC12 cells, where sAC is expressed at high levels
(26). To do so, we used a variant of the genetically encoded
FRET-based biosensor A-kinase activity reporter 4 (AKAR4)
targeted to the plasma membrane with the farnesylation lipid
modification sequence derived from K-Ras (AKAR4-Kras)
(Fig. 1A) (3). This biosensor detects an increase in plasma
membrane-localized PKA activity as an increase in the yellow/
cyan emission ratio, and the maximal response of AKAR4-
Kras, as determined by simultaneous addition of the tmAC
activator forskolin (Fsk) and the general PDE inhibitor
3-isobutyl-1-methylxanthine (IBMX), is 14.5% ? 2.9% (n ? 6)
[mean ? standard error of the mean (SEM) (n ? number of
cells)] (Fig. 1B, open squares). Since sAC expression is high at
the plasma membrane in PC12 cells (26), activation of TrkA
with NGF and subsequent sAC activation were expected to
induce robust PKA activity near the plasma membrane. In-
deed, when PC12 cells expressing AKAR4-Kras were treated
with NGF, there was rapid and robust PKA activation, as is
indicated by an increase in the yellow/cyan emission ratio of
12.6% ? 1.4% (n ? 11) (Fig. 1C, purple diamonds, and D,
top). We next wanted to examine the effect of EGF on PKA
activity in PC12 cells. Since EGF was shown not to increase
cAMP or activate PKA in PC12 cells (14, 36), it was unex-
pected when EGF induced an AKAR4-Kras response of
9.1% ? 1.2% (n ? 12) (Fig. 1E, purple diamonds, and F, top).
However, in contrast to the sustained PKA activity observed
with NGF stimulation, EGF-induced PKA activity at the
plasma membrane was transient, reversing back to basal levels
within 15 min following treatment (Fig. 1E, purple diamonds,
F, and G). Together these observations identify NGF and EGF
as potent activators of PKA at the plasma membrane, although
they induce temporally distinct patterns of PKA activity.
We next wanted to examine the effect that both GFs had on
cytosolic PKA activity by using an AKAR4 variant targeted to
the cytosol with a nuclear export signal (NES) (AKAR4-NES),
which displays a maximal yellow/cyan emission ratio increase
of 51.3% ? 1.8% (n ? 5) in response to Fsk and IBMX in
4064HERBST ET AL.MOL. CELL. BIOL.
PC12 cells (Fig. 1A and B, solid squares). Surprisingly, in cells
expressing AKAR4-NES, NGF treatment elicited a negligible
AKAR4-NES response (0.7% ? 0.2%; n ? 18), suggesting that
NGF is incapable of activating cytosolic PKA (Fig. 1C and D).
Similarly, no cytosolic PKA activity was observed when cells
expressing AKAR4-NES were treated with EGF (Fig. 1E and
F), showing that EGF-induced PKA activity is also subject to
precise spatial compartmentalization. Since this trend of GF-
stimulated plasma membrane-compartmentalized PKA activ-
ity was observed with both NGF and EGF, these observations
point to a molecular signaling barrier that is responsible for
localizing GF-induced PKA activity to the plasma membrane
while restricting the activation of cytosolic PKA.
PDE3 is a major isoform in PC12 cells. Since PDEs are
negative regulators of PKA signaling and are known to serve as
a signaling barrier or sink to help establish cAMP gradients in
cells (1), we investigated if PDEs contribute to the plasma
membrane (PM)-compartmentalized PKA activity down-
stream of growth factor receptor activation. When cells ex-
pressing AKAR4-Kras or AKAR4-NES were treated with the
general PDE inhibitor IBMX, an increase in the yellow/cyan
emission ratio of 18.4% ? 2.4% (n ? 13) and 27.3% ? 6.3%
(n ? 9), respectively, was observed (Fig. 2A to C, black),
indicating that PDEs play a role in regulating basal cAMP
levels and thus PKA activity in PC12 cells. Next, we used the
PDE3 isoform-selective inhibitor milrinone and the PDE4-
selective inhibitor rolipram to examine the roles of PDE3 and
PDE4 on regulating PKA activity in these cells. First, at both
the membrane and cytosol, PDE3 inhibition with milrinone
activated PKA to levels comparable to those induced by
IBMX, with responses of 13.1% ? 0.9% (n ? 16) for AKAR4-
Kras and 23.7% ? 4.2% (n ? 7) for AKAR4-NES (Fig. 2A to
C, gray). Conversely, cells that were treated with rolipram
showed negligible changes in PKA activity at the PM (3.4% ?
0.7%; n ? 9) and in the cytosol (1.9% ? 0.5%; n ? 15) (Fig.
2A to C). Using a cytosolic biosensor for cAMP levels, Epac1-
camps (16, 18), we observed effects of PDE inhibition on
cAMP levels similar to those that were observed on PKA
activity, specifically that IBMX and milrinone generate similar
levels of cAMP accumulation while rolipram generates less
cAMP (Fig. 2D). In agreement with previous studies (34),
together these data suggest that PDE3, in contrast to PDE4, is
a major isoform in PC12 cells and regulates basal PKA activity
in these cells.
Next, to confirm the expression of PDE3 in PC12 cells, we
performed immunoblot analysis using a PDE3B antibody.
Since PDE3B is known to be a membrane-associated protein
(2), we sought to quantify the relative expression of PDE3B in
FIG. 1. GF-induced PKA activity is spatiotemporally regulated in PC12 cells. (A) Schematic representation of AKAR4-Kras and AKAR4-NES.
(B) PC12 cells expressing AKAR4-NES (n ? 3) or AKAR4-Kras (n ? 6) were treated with Fsk (50 ?M) plus IBMX (100 ?M). Y/C, yellow/cyan
emission ratio. (C) Time courses depicting the NGF (200 ng/ml)-induced responses of AKAR4-Kras (n ? 5) and AKAR4-NES (n ? 5). (D) YFP
images and ratiometric images of AKAR4-Kras (top) and AKAR4-NES (bottom) before NGF addition (0 min) and at 5 min and 12.5 min after
NGF addition. (E) Time courses depicting the EGF (100 ng/ml)-induced responses of AKAR4-Kras (n ? 6) and AKAR4-NES (n ? 3). (F) YFP
images and ratiometric images of AKAR4-Kras (top) and AKAR4-NES (bottom) before EGF addition (0 min) and at 5 min and 12.5 min after
EGF addition. (G) Bar graph depicting the AKAR4-Kras responses at 2.5, 5, 10, and 12.5 min following NGF (n ? 11) or EGF (n ? 12) treatment.
n.s, no statistically significant difference between indicated treatments;**, P ? 0.01;***, P ? 0.005. All data are shown as means ? SEMs.
VOL. 31, 2011 SPATIOTEMPORAL REGULATION OF GF-MEDIATED PKA ACTIVITY4065
the cytosolic and membrane compartments of PC12 cells. To
do so, we separated the cytosolic and membrane fractions of
PC12 cells via centrifugation and subsequently separated the
proteins present in each fraction by SDS-PAGE. When both
fractions were probed with an anti-tubulin antibody, the signal
was observed predominantly in the cytosolic fraction, indica-
tive of clean separation of the membrane from the cytosol. In
contrast, when both fractions were probed for PDE3B, we
observed 89.6% ? 2.6% of the total PDE3B in the membrane
fraction (Fig. 2E). These data signify that PDE3B not only is
present in PC12 cells but also is primarily membrane associ-
PDE3 is a critical regulator of the GF-stimulated spatio-
temporal dynamics of PKA activity. We next sought to inves-
tigate whether PDE3 contributes to the spatiotemporally reg-
ulated PKA activities downstream of TrkA and EGFR
activation. To begin, we examined if PDE3 contributes to the
spatial compartmentalization of GF-stimulated PKA to the
plasma membrane. To do so, we concurrently treated cells
expressing AKAR4-NES with GF and a low dose of milrinone
(5 ?M). Notably, while 1 ?M milrinone generates a maximal
response from AKAR4-Kras (data not shown), 5 ?M milri-
none induces a negligible response of 0.7% ? 0.6% from
AKAR4-NES, indicating that this dose of milrinone does not
activate cytosolic PKA (Fig. 3A, gray). Importantly, this obser-
vation is consistent with the notion that PDE3, while playing a
role in regulating PKA activity at the plasma membrane in the
basal state, is potent at restricting PKA activation in the cyto-
sol. When NGF and a low dose of milrinone (5 ?M) were
simultaneously added to cells, an increase in PKA activity was
observed (2.2% ? 0.3%; n ? 12) (Fig. 3A and B). Similarly, an
AKAR4-NES response of 2.6% ? 0.4% (n ? 6) was detected
when cells were concurrently treated with EGF and low-dose
milrinone (Fig. 3A and C). Similar trends were observed using
Epac1-camps; specifically, EGF and NGF did not produce
detectable levels of cytosolic cAMP unless PDE3 was partially
inhibited (Fig. 3D), and these trends were confirmed using
cAMP enzyme immunoassays (EIA) (data not shown). To-
gether these data suggest that cAMP produced by TrkA and
EGFR activation is rapidly degraded by PDE3 and is only
capable of diffusing into the cytosol when PDE3 is partially
inhibited. In contrast to cells cotreated with GF and a low dose
of milrinone, those cells that were simultaneously treated with
GF and a submaximal dose of rolipram (0.5 ?M) did not
display detectable cytosolic PKA activity (Fig. 3E) or cAMP
FIG. 2. PDE3, but not PDE4, regulates both PM and cytosolic
PKA activity in PC12 cells. (A) PC12 cells expressing AKAR4-Kras
were treated with 100 ?M IBMX (n ? 4), 10 ?M milrinone (Mil) (n ?
5), or 1 ?M rolipram (Roli) (open circles; n ? 5), and PKA activity was
monitored. (B) Cytosolic PKA activity was detected in cells expressing
AKAR4-NES treated with 100 ?M IBMX (n ? 4), 10 ?M Mil (n ? 5),
or 1 ?M Roli (n ? 6). (C) Responses of AKAR4-Kras and AKAR4-
NES induced by IBMX (Kras [n ? 13], NES [n ? 9]), Mil (Kras [n ?
16], NES [n ? 7]), and Roli (Kras [n ? 9], NES [n ? 15]). (D) Bar
graph depicting the cAMP concentration ([cAMP]) induced by various
treatments, as determined by Epac1-camps [50 nM Fsk (n ? 11), 100
?M IBMX (n ? 8), 10 ?M Mil (n ? 15), 1 ?M Roli (n ? 11)]. (E)
PC12 cells were separated into cytosolic (C) and membrane (M) frac-
tions by centrifugation, and each fraction was separated by SDS-
PAGE. Immunoblot analysis (top) is representative of one fraction-
ation, and quantification of the relative expression of PDE3B in each
fraction (bottom) is representative of four independent fractionation
experiments and subsequent immunoblot analysis. Tubulin serves as a
control for clean separation of the membrane and cytosolic fractions.
All data are presented as means ? SEMs; “n.s.” indicates no statisti-
cally significant difference between indicated treatments.**, P ? 0.01;
***, P ? 1E?05.
4066 HERBST ET AL.MOL. CELL. BIOL.
increases shown by cAMP EIA (data not shown). Taken with
the observation that NGF and EGF are robust activators of
PKA at the plasma membrane, the finding that partial phar-
macological inhibition of PDE3 is necessary for both NGF and
EGF to activate cytosolic PKA suggests that PDE3 functions to
restrict the diffusion of plasma membrane-generated cAMP
into the cytosol, where it could activate cytosolic PKA. As
inhibition of PDE3 also alters the kinetics of EGF-induced
PKA activity at the plasma membrane (Fig. 3F), these findings
also show that pharmacological manipulation of PDE3 activity
can be used as a tool to disrupt the spatiotemporal regulation
of GF-mediated PKA signaling.
cAMP-mediated signaling regulates GF-stimulated cytoso-
lic ERK activity. Given that cAMP and ERK signaling are
intricately connected in PC12 cells (28), we next investigated
how PDE3 and other components of cAMP signaling influence
GF-induced ERK activity. To this end, we used the ERK
activity reporter (EKAR), a FRET-based biosensor that spe-
cifically detects ERK activity as an increase in the yellow/cyan
emission ratio (7), and first tested the effect of PKA-mediated
signaling on the regulation of GF-stimulated ERK activity. In
PC12 cells expressing the cytosolic EKAR (EKARcyto), NGF
treatment results in a 23.6% ? 1.3% (n ? 8) increase in the
yellow/cyan emission ratio, with a time to reach half-maximal
activation (t1/2) of 5.0 ? 0.1 min (mean ? SEM) (Fig. 4A and
B). EGF generated a similar response, with a maximal emis-
sion ratio increase of 19.8% ? 0.6% (n ? 23) and a t1/2of 4.6 ?
0.2 min (Fig. 4A and C). In both cases, the GF-induced ERK
activity was abolished when cells were pretreated with 20 ?M
MEK inhibitor U0126 (data not shown). To test the effect of
PKA on regulating GF-induced ERK activity, cells expressing
EKARcytowere pretreated with the relatively selective PKA
inhibitor H89 for 10 min, followed by treatment with either
NGF or EGF. In both cases, inhibition of PKA significantly
slowed the onset of ERK activation induced by NGF and EGF,
increasing the t1/2values to 10.0 ? 1.2 min (n ? 14) and 5.9 ?
0.4 min (n ? 9), respectively (Fig. 4A to C). As similar in-
creases in t1/2were observed in cells expressing the PKA pep-
tide inhibitor PKI? (Fig. 4A to C), these findings show that
PKA plays a role in activating ERK following activation of
both TrkA and EGFR, although the effect is more pronounced
in the case of TrkA activation. Furthermore, disruption of
FIG. 3. GFs activate cytosolic PKA when PDE3 is partially inhibited. (A) Bar graph depicting the negligible AKAR4-NES response induced
by GF alone (white bars; NGF [n ? 4] and EGF [n ? 12]) and a subsaturating dose of milrinone (5 ?M) (gray bars; n ? 10). In contrast, when
cells are simultaneously treated with GF and 5 ?M milrinone (black bars; NGF [n ? 12] and EGF [n ? 6]), an increase in cytosolic PKA activity
is observed. (B) Time course of PC12 cells expressing AKAR4-NES treated with NGF alone (n ? 5) or NGF plus 5 ?M milrinone (submil n ?
3). (C) Time course depicting the AKAR4-NES response of cells treated with EGF alone (n ? 3) or EGF plus 5 ?M milrinone (n ? 3). (D) Bar
graph depicting the cAMP concentration induced by various treatments, as determined by the response of Epac1-camps (NGF [n ? 8], EGF [n ?
7], 5 ?M Mil [n ? 15], NGF plus 5 ?M Mil [n ? 14], and EGF plus 5 ?M Mil [n ? 10]). (E) A submaximal dose of rolipram (0.5 ?M) does not
activate cytosolic PKA in the absence (gray bars; n ? 5) or presence (black bars; NGF [n ? 11] and EGF [n ? 9]) of GF. (F) The EGF-induced
PKA activity at the plasma membrane (n ? 12) is sustained in the presence of PDE3 inhibition with milrinone (10 ?M) (n ? 8) but not in the
presence of PDE4 inhibition with rolipram (1 ?M) (n ? 6). All data are shown as means ? SEMs.*, P ? 0.05.
VOL. 31, 2011SPATIOTEMPORAL REGULATION OF GF-MEDIATED PKA ACTIVITY4067
FIG. 4. cAMP-mediated signaling regulates ERK activity in PC12 cells. (A) Bar graph depicting the time to reach half-maximal activation
[(t(1/2)] of EKARcyto in response to various treatments (NGF alone [n ? 12], NGF after H89 pretreatment [n ? 14], NGF in the presence of
PKI? expression [n ? 5], EGF alone [n ? 23], EGF after H89 pretreatment [n ? 13], and EGF in the presence of PKI? expression [n ? 5]).
(B) Time courses of PC12 cells expressing EKARcytotreated with NGF (Cntl; n ? 8), NGF after 10 ?M H89 pretreatment (n ? 7), or NGF in
the presence of PKI? expression (n ? 5). (C) Time courses depicting the EKARcytoresponse to EGF (cntl; n ? 8), EGF after H89 pretreatment
4068HERBST ET AL.MOL. CELL. BIOL.
PKA signaling by addition of St-Ht31, a peptide which disrupts
PKA/AKAP interactions (12), decreases the magnitude of
both NGF- and EGF-induced ERK activity and significantly
delays the onset of NGF-induced ERK activity (Fig. 4D and
E). These data not only further establish a role of PKA in
regulating GF-induced ERK activity but also suggest that this
regulation is achieved through a pool of PKA anchored by
Next, to examine the effect of sAC on GF-induced ERK
activity, cells were pretreated with a sAC inhibitor, KH7, and
subsequently treated with GF. The onset of ERK activity was
delayed in the case of NGF-treated cells (t1/2? 6.12 ? 0.4 min;
(n ? 4), and EGF in the presence of PKI? expression (n ? 3). (D and E) NGF (D)- and EGF (E)-induced time courses of ERK activity in cells
in which PKA anchoring was disrupted via pretreatment with Ht31 (1 ?M). (F) Bar graph depicting the time to reach half-maximal activation of
EKARcytoin response to various treatments (NGF alone [n ? 12], NGF after KH7 pretreatment [n ? 14], EGF alone [n ? 23], and EGF after
KH7 pretreatment [n ? 13]). (G) Time courses of PC12 cells expressing EKARcytotreated with NGF (Cntl; n ? 8) or NGF after 30 ?M KH7
pretreatment (n ? 7). (H) Time courses depicting the EKARcytoresponse to EGF (Cntl; n ? 8) and EGF after KH7 pretreatment (n ? 7). (I)
PC12 cells were pretreated with vehicle, 30 ?M KH7 (K), or 10 ?M H89 (H) for 20 min and subsequently treated with NGF (N) or EGF (E) for
5 min or 15 min. Control cells (Cntl) were treated with vehicle only. Immunoblots of p-ERK1/2 and tubulin (loading control) represent a single
experiment. (J) Quantifications of p-ERK1/2 over tubulin for the NGF-treated samples are plotted and normalized to the vehicle-treated sample
at each time point (n ? 3 to 5 independent experiments). (K) Quantifications of p-ERK1/2 over tubulin for the EGF-treated samples are plotted
and normalized to the vehicle-treated sample at each time point (n ? 3 to 5 independent experiments). All imaging and immunoblot data are
shown as means ? SEMs.*, P ? 0.05;**, P ? 0.01;***, P ? 0.001; n.d., not detected.
FIG. 5. PDE3, but not PDE4, regulates ERK activity in PC12 cells. (A) Bar graph depicting the t1/2s of various treatments (NGF alone [n ?
12], NGF after milrinone [M] pretreatment [n ? 14], NGF after pretreatment with 1 ?M rolipram [R] [n ? 7], EGF alone [n ? 23], EGF after
milrinone pretreatment [n ? 12], and EGF after rolipram pretreatment [n ? 16]). (B) Time courses depicting the EKARcytoresponse from cells
treated with NGF (Cntl; n ? 8) and NGF after pretreatment with 10 ?M milrinone (Mil; n ? 8). (C) Time courses depicting the EKARcyto
response of cells treated with EGF (Cntl; n ? 8) and EGF after pretreatment with milrinone (n ? 6). (D) PC12 cells were pretreated with vehicle
or 10 ?M milrinone (M) for 20 min and subsequently treated with NGF or EGF for 2.5 min, 5 min, or 15 min. Control cells (Cntl) were treated
with vehicle only. Immunoblots of p-ERK1/2 and tubulin represent a single experiment. (E) Quantifications of p-ERK1/2 over tubulin for the
NGF-treated samples are plotted and normalized to the vehicle-treated sample at each time point (n ? 3 independent experiments). (F) Quan-
tifications of p-ERK1/2 over tubulin for the EGF-treated samples are plotted and normalized to the vehicle-treated sample at each time point (n
? 3 independent experiments). All imaging and immunoblot data are shown as means ? SEMs.*, P ? 0.05;**, P ? 0.01; n.d., not detected.
VOL. 31, 2011SPATIOTEMPORAL REGULATION OF GF-MEDIATED PKA ACTIVITY 4069
FIG. 6. PDE3 inhibition extends the duration of EGF-induced nuclear ERK in a PKA-dependent manner. All experiments are done in PC12
cells expressing EKARnuclear. (A) YFP direct (left) and ratiometric images depicting differential GF-induced nuclear ERK activities in the presence
and absence of PDE3 inhibitor (Mil) and/or PKA inhibitor (H89). (B) Graphical representation of the time needed for nuclear ERK activity to
reduce to 50% [t1/2(Rev)]. (C) NGF-induced nuclear ERK activity (n ? 9) is more sustained than EGF-induced nuclear ERK activity (n ? 10).
(D) Time course of NGF-induced nuclear ERK activity in the presence of H89 (n ? 8) and PKI? (n ? 5) expression. (E) tmAC activation with
increasing doses of Fsk extends the duration of EGF-stimulated ERK activity in the nucleus (0.05 ?M Fsk, n ? 8; 0.5 ?M Fsk, n ? 9; 5.0 ?M Fsk,
4070 HERBST ET AL.MOL. CELL. BIOL.
n ? 10), whereas sAC inhibition had no effect on EGF-stim-
ulated ERK activity (t1/2? 4.1 ? 0.2 min; n ? 13) (Fig. 4F to
H). Conversely, cells which were pretreated with 10 ?M MDL-
12330A, a tmAC inhibitor, showed a dramatic reduction in
EGF-stimulated ERK activity compared to cells which were
not pretreated (data not shown). Together these findings sug-
gest that both NGF- and EGF-induced activation of ERK is
dependent on cellular levels of cAMP, although the effects of
the two GFs may be differentially regulated by sAC and tmAC.
Live-cell imaging with FRET-based kinase activity reporters
is a sensitive method to monitor the spatiotemporal regulation
of kinase activity in real time, but in some cases, these probes
are so sensitive that they can be easily saturated when a kinase
is robustly activated, making it difficult to discern discrepancies
in the magnitude of kinase activity. Since we observed a slight
reduction in the GF-induced EKAR response in the presence
of PKA inhibition with H89 and PKI? (Fig. 4B and C), we
sought to further investigate the effect of KH7 and H89 on
GF-stimulated ERK activity with immunoblot analysis, a tech-
nique which is less prone to signal saturation. Therefore, to
monitor the effect of sAC and PKA inhibition on the magni-
tude of GF-stimulated ERK activity, we monitored the levels
of phosphorylated ERK1/2 (p-ERK) in a population of PC12
cells in response to various treatments. In doing so, we first
showed that inhibition of sAC by pretreatment for 20 min with
KH7 significantly reduces the levels of p-ERK induced by NGF
5 min after treatment but had little effect on p-ERK levels 15
min post-NGF treatment (Fig. 4I and J), confirming that NGF-
stimulated ERK activity is dependent on sAC activity, espe-
cially at earlier time points. Similarly, we also showed that
inhibition of PKA with H89 dramatically reduced the levels of
p-ERK induced by NGF at 5 min following NGF treatment,
but at 15 min post-NGF treatment, there was no detectable
difference in p-ERK levels in the presence or absence of H89
treatment, confirming our findings with EKAR imaging (Fig.
4I and J).
In the case of EGF, we found that sAC inhibition with KH7
decreased the amplitude of EGF-stimulated p-ERK levels 5
min after EGF treatment (Fig. 4I and K). This finding is in
contrast to what was observed in imaging experiments using
EKAR, and this discrepancy is likely due to the difference in
these two assays in which EKAR is saturated more easily,
making it more difficult to detect changes in the magnitude of
ERK signaling. However, when taken with the observation that
the inhibition of tmAC can dramatically reduce the magnitude
of EGF-induced ERK activity (data not shown), the observa-
tion that sAC inhibition affects the levels of EGF-induced
p-ERK suggests that EGF-induced ERK activity is largely de-
pendent on the levels of cAMP in the cell. Finally, we also
showed that inhibition of PKA decreases the levels of p-ERK
5 min following EGF treatment (Fig. 4I and K). Regardless of
the pretreatment, p-ERK levels were undetectable 15 min af-
ter EGF treatment. Together with our imaging data, the results
of the immunoblot analysis confirm that cAMP/PKA signaling
is a critical factor in the regulation of GF-induced ERK
Next, to test the involvement of PDE3 in regulating GF-
induced ERK activity, we pretreated cells with 10 ?M PDE3
inhibitor milrinone for 10 min and then stimulated cells with
either of the GFs. Under these conditions, there is a reduction
in the onset of both NGF- and EGF-stimulated ERK activity,
with t1/2s of 3.6 ? 0.3 min (n ? 14) and 3.7 ? 0.3 min (n ? 12),
respectively, compared to those of cells that were not pre-
treated with milrinone (Fig. 5A to C), and the EKAR re-
sponses were abolished in the presence of MEK inhibition
(data not shown). These findings indicate that PDE3, via reg-
ulation of GF-stimulated cAMP and PKA dynamics, also reg-
ulates GF-stimulated ERK activity. In agreement with this, in
some cells, pharmacological inhibition of PDE3 alone was suf-
ficient to activate ERK (data not shown), but these cells were
not included in the analysis. Furthermore, in contrast to cells
pretreated with milrinone, those that were pretreated with
rolipram did not show a change in GF-stimulated ERK activity
(Fig. 5A), consistent with the finding that PDE4 inhibition
does not potently activate PKA in these cells.
Again, we sought to confirm our imaging data and investi-
gate differences in the magnitude of ERK signaling using im-
munoblot analysis. In the case of NGF, we observed that in-
hibition of PDE3 resulted in significantly greater levels of
p-ERK at 2.5 min following NGF treatment (Fig. 5D and E),
similar to that observed with EKAR imaging. After NGF treat-
ment for 5 or 15 min, however, the discrepancy in p-ERK levels
in the presence or absence of milrinone was not as pronounced
(Fig. 5D and E), again in agreement with our imaging data. We
also confirmed that EGF-induced p-ERK levels are enhanced
by inhibition of PDE3 at 2.5 min following EGF treatment, and
PDE3 inhibition had less of an effect on p-ERK levels 5 min
after EGF treatment (Fig. 5D and F). Together with the im-
aging data, the immunoblot data show that cAMP, along with
PKA and PDE3, are critical regulators of ERK signaling in
PC12 cells, such that subtle increases in intracellular cAMP
can prime both the NGF- and EGF-mediated signaling to
more rapidly activate ERK.
Disruption of the spatiotemporal regulation of PKA activity
alters EGF-stimulated ERK activity in the nucleus. We next
wanted to investigate if PDE3 and spatiotemporally controlled
PKA activity contribute to the specific growth factor signaling
observed in PC12 cells by monitoring the effect of PDE3 in-
hibitor on the duration of nuclear ERK activity, where we have
found it easiest to discern discrepancies between the duration
of ERK activity induced by EGF and NGF (data not shown).
To begin, we used a nucleus-localized EKAR (EKARnuclear) as
n ? 10; 50 ?M Fsk, n ? 7. (F) Bar graph depicting the increase in reversal of nuclear ERK activity [t1/2(Rev)] following various treatments (EGF
plus the following doses of Fsk: 0.05 ?M, n ? 16; 0.5 ?M, n ? 17; 5.0 ?M, n ? 10; 50 ?M Fsk, n ? 7). (G) PDE3 inhibition with 10 ?M Mil (n ?
7) extends the duration of EGF-stimulated ERK activity. (H and I) 100 ?M IBMX increases the duration of EGF-stimulated nuclear ERK activity
(n ? 8) (H), while 1 ?M Roli does not (n ? 7) (I). (J) In the presence of milrinone and inhibition of PKA through H89 pretreatment (n ? 8)
or PKI? expression (n ? 5), there is no increase in the duration of EGF-induced nuclear ERK activity. All data are shown as means ? SEMs. ??,
P ? 0.01 compared to EGF.**, P ? 0.01 compared to NGF.
VOL. 31, 2011 SPATIOTEMPORAL REGULATION OF GF-MEDIATED PKA ACTIVITY4071
a tool to monitor the differential growth factor signaling that
results from NGF and EGF. As expected, in cells expressing
EKARnuclear, we observed sustained and transient durations of
nuclear ERK activity in response to NGF and EGF, respec-
tively, with the corresponding time to reverse to half-maximal
ERK activity [t1/2(Rev)] of 53.1 ? 4.5 min (n ? 19) after NGF
addition and 14.6 ? 0.9 min (n ? 20) after EGF addition (Fig.
6A to C), and the kinetics of ERK activity observed with
EKARnuclearcorrespond with translocation of ERK into and
out of the nucleus (data not shown). The involvement of PKA
in promoting the sustained phase of NGF-stimulated ERK
activity was tested by simultaneously treating cells with NGF
and the PKA inhibitor H89. Under these conditions, a more
transient EKARnuclearresponse was observed, with a t1/2(Rev)
value of 30.3 ? 3.8 min (n ? 9) (Fig. 6D, orange). As a similar
effect was observed in cells expressing PKI? (Fig. 6D, green),
these observations confirm that PKA is at least partially re-
sponsible for sustaining ERK activity as a result of NGF treat-
ment. Also in support of a role of cAMP/PKA signaling in
promoting sustained ERK activity, compared to cells treated
with EGF alone, cells treated with EGF and increasing doses
of Fsk, which correlate with increased levels of cAMP pro-
duced (data not shown), show a dose-dependent increase in
the duration of nuclear ERK activity (Fig. 6E and F). Specif-
ically, EGF in conjunction with 0.05, 0.5, 5.0, or 50 ?M Fsk
show t1/2(Rev)values of 19.1 ? 1.2 min (n ? 16), 23.8 ? 2.6 min
(n ? 17), 27.4 ? 1.1 min (n ? 10), or 32.4 ? 1.9 min (n ? 7),
respectively (Fig. 6B, E, and F). While these data are in agree-
ment with the early observation that cAMP in conjunction with
EGF can cause sustained ERK activation on a global level
(35), EKARnuclearallows for the specific detection of nuclear
ERK activity, in which we have observed more dramatic tem-
poral differences in NGF- versus EGF-stimulated ERK activity
relative to the cytosol (data not shown). Since it is clear that a
high concentration of cAMP, like that generated by Fsk (Fig.
2D), can sustain EGF-stimulated ERK activity in the nucleus,
we wanted to test the effect of PDE3 inhibition alone, which
induces less cAMP than Fsk at tested doses (Fig. 2D) yet
perturbs the compartmentalization of PKA activity (Fig. 3).
When cells expressing EKARnuclearwere stimulated with EGF
in conjunction with 10 ?M milrinone, there was a significantly
longer t1/2(Rev)(26.5 ? 3.0 min; n ? 20) compared to that of
cells treated only with EGF (Fig. 6A, B, and G), and a similar
effect was observed with IBMX (Fig. 6B and H) but not rolip-
ram (Fig. 6B and I), illustrating a role for PDE3 in the regu-
lation of EGF-stimulated nuclear ERK activity. We did not,
however, observe an extended duration of NGF-stimulated
nuclear ERK activity in the presence of milrinone or 50 ?M
Fsk, suggesting that NGF alone can achieve the maximal du-
ration of nuclear ERK activity (data not shown). We further
showed that the extended duration of EGF-induced nuclear
ERK activity that results from PDE3 inhibition is largely de-
pendent on PKA activity since simultaneous treatment with
EGF and milrinone in the presence of H89 did not result in
sustained nuclear ERK activity with a t1/2(Rev)of 14.3 ? 1.0
min (n ? 8) (Fig. 6A, B, and J, green), and a similar trend was
observed in cells expressing PKI? (Fig. 6J, pink). Together
with our observation that PDE3 regulates the plasma mem-
brane compartmentalization of GF-stimulated PKA activity
and the duration of EGF-stimulated PKA activity in that locale
(Fig. 3), our studies with EKARnuclearshow that PDE3 con-
trols the spatiotemporal patterns of EGF-stimulated PKA ac-
tivity and in turn acts as a critical regulator of nuclear ERK
activity in PC12 cells.
To test if the duration of nuclear ERK activity induced by
various treatments correlates with neurite extension, we
treated PC12 cells expressing a plasma membrane-targeted
YFP for 3 days with various treatments and monitored their
effects on neurite outgrowth. Membrane-targeted YFP is used
to highlight the cell membrane and facilitate the quantification
of neurite lengths. Consistent with the literature, we see that
NGF induces significant neurite outgrowth (neurites were de-
fined as processes with lengths greater than or equal to the
length of the cell body), whereas EGF treatment does not. We
also observe that EGF plus 5 ?M Fsk induces neurite out-
growth (35), although the effect on neurite outgrowth is not as
robust as that of NGF treatment, in terms of number of neu-
rites or neurite length. Also consistent with the literature, we
see that PKA inhibition with H89 does not reverse the effect of
NGF on neurite outgrowth, whereas the effect of EGF plus Fsk
is somewhat reversed upon addition of H89 (35). In the case of
EGF plus milrinone, IBMX, H89, or milrinone and H89, we do
not see significant effects on neurite outgrowth compared to
cells treated with EGF alone (Fig. 7). Interestingly, our studies
with EKARnuclear(Fig. 6) clearly show that the following treat-
ments have indistinguishable effects on the duration of nuclear
ERK activity: NGF plus H89, EGF plus milrinone, EGF plus
IBMX, and EGF plus 5 ?M Fsk. On the other hand, it is clear
that these treatments show different effects on neurite out-
growth, specifically that NGF plus H89 and EGF plus Fsk
induce neurite outgrowth, whereas EGF plus milrinone and
EGF plus IBMX do not. These data demonstrate the complex-
ity of the integrated controls by the cAMP and ERK signaling
systems on cellular processes and suggest that additional sig-
naling players are likely involved in regulating the differentia-
tion process in PC12 cells. Future studies are needed to iden-
tify these signaling molecules and to characterize the role that
the differential spatiotemporal activity patterns of GF-induced
PKA activities have on these signaling molecules and their role
in the differentiation of PC12 cells.
In this study, the utilization of FRET-based biosensors,
which have proven to be powerful tools for analyzing dynamic
and spatially compartmentalized signaling activities (8, 38), has
led to a few new observations. While it is known that NGF
activates sAC to produce cAMP in PC12 cells (26) and that the
activation of Rap1 and ERK via NGF treatment is PKA de-
pendent (19), it has been difficult to directly detect the activa-
tion of PKA by NGF treatment (19, 36). One possible reason
is that the averaged signals obtained when the NGF-induced
PKA activity was monitored in whole cells result in low sensi-
tivity. In contrast, localized FRET-based biosensors allow for
the local detection of kinase activity and have led to the ob-
servation that NGF is indeed a robust activator of PKA activity
at the plasma membrane but not in the cytosol. Moreover,
using the targeted FRET-based biosensor approach, we were
able to detect EGF-induced PKA activity in PC12 cells, un-
covering a previously unknown mode of activation of PKA by
4072 HERBST ET AL.MOL. CELL. BIOL.
EGF in this cell model. In the same vein, subcellularly targeted
ERK biosensors revealed localized ERK activity patterns stim-
ulated by different GFs. Specifically, we observed larger dis-
crepancies in the transient versus sustained ERK activity in-
duced by EGF and NGF in the nucleus than in the cytoplasm,
suggesting that the temporally controlled ERK activities by
different GFs can be further amplified by spatial constraints
One critical finding from our study is the compartmentaliza-
tion of GF-stimulated PKA activity to the plasma membrane.
Previous studies have identified the plasma membrane to be a
unique site of GF-regulated signaling. The signaling sensitivity
appears to be high at this location, such that even low-level
input signals can activate the Raf-MEK-ERK pathway (6, 17,
33). Since PKA is an upstream activator of Raf, our finding
that GF-induced PKA activity is localized to the plasma mem-
brane suggests a PKA-dependent mechanism for this observa-
tion and further supports the role of the plasma membrane as
an important signaling platform in PC12 cells. While we have
identified PDE3 to be critical in restricting NGF- and EGF-
induced PKA activity to the plasma membrane, it is likely that
there are other molecular players that help to confer this
precise compartmentalization. For instance, our observation
that disruption of PKA/AKAP interactions with Ht31 de-
creases the magnitude and onset of GF-stimulated ERK activ-
ity (Fig. 4D and E) suggests that specific AKAPs (25) could
play a role in localizing the molecular machinery involved in
PKA and ERK cross talk to the plasma membrane to maintain
Interestingly, although both EGF and NGF stimulate PKA
activities that are restricted to the plasma membrane, the tem-
poral patterns of the stimulated activities differ. One potential
mechanism may be via differential regulation involving differ-
ent sources of cAMP. Additional factors may also contribute.
For instance, since EGFR is known to desensitize shortly after
activation, it is possible that activation of tmACs via EGFR is
FIG. 7. The duration of nuclear ERK activity does not precisely correlate with neurite outgrowth. PC12 cells expressing a plasma membrane-
targeted YFP were treated for 3 days as indicated, and the number of cells expressing neurites (defined as a cellular extension longer than the
length of the cell body) was quantified. n ? 25 to 105 cells per treatment. Representative images for each treatment are shown.
VOL. 31, 2011 SPATIOTEMPORAL REGULATION OF GF-MEDIATED PKA ACTIVITY4073
transient due to the internalization of the receptor and desen-
sitization of the signaling pathway (20, 31). It is also possible
that activated EGFR and TrkA could differentially activate
other signaling molecules, such as protein kinase C (PKC)
(22), which could contribute to the transient and sustained
EGF- and NGF-stimulated PKA activity at the plasma mem-
brane. Future studies will focus on further elucidating the
underlying molecular mechanisms which control the specific
temporal patterns of EGF- and NGF-stimulated PKA activity
at the plasma membrane.
While several signaling components are known to be in-
volved in the temporal regulation of ERK, thereby influencing
the cell fate specification achieved by EGF and NGF in PC12
cells (14, 15, 20, 22, 23, 30, 34), our study revealed the precise
spatiotemporal regulation of GF-stimulated PKA activity,
which is tightly controlled by PDE3, as a new level of regula-
tion underlying the growth factor signaling specificity in these
cells. Specifically, while NGF and EGF stimulate sustained and
transient nuclear ERK responses, respectively, pharmacologi-
cal inhibition of PDE3 resulted in a nearly a 2-fold increase in
the duration of EGF-stimulated nuclear ERK activity. Since
the amount of cAMP produced by PDE3 inhibition is small
(Fig. 2D), this effect is likely to be mediated by the altered
spatiotemporal pattern of PKA activity. As PKA is an estab-
lished upstream activator of Rap1 and Raf, both previously
identified to regulate the differential responses of EGF and
NGF at the level of ERK activation (27, 23), it will be inter-
esting to monitor the effect of altering PKA spatiotemporal
dynamics has on these molecules. Importantly, while our find-
ings cannot distinguish whether the nearly 2-fold increase in
the duration of nuclear ERK activity in the presence of PDE3
inhibition is due to active cytosolic PKA or sustained PKA
activity at the plasma membrane, they illustrate the importance
of spatiotemporally regulated PKA activity in contributing to
the control of specific signaling effects of EGF and NGF.
However, at the functional level, we have shown that EGF in
the presence of the tmAC activator and EGF in the presence
of the PDE3 inhibitor, two conditions that cause similar effects
on the duration of nuclear ERK activity, do not have the same
effect on neurite outgrowth (Fig. 6 and 7). This finding that the
duration of nuclear ERK activity do not precisely correlate
with the induction of neurite outgrowth illustrates the com-
plexity of integrated control by the cAMP and ERK signaling
systems on cell differentiation. The distinct signaling activity
patterns and gene expression profiles induced by NGF and
EGF will be further analyzed in future studies.
Together, the findings in this study have led to an enhanced
understanding of the molecular mechanisms underlying the
GF signaling specificity in this classical cell model system.
Furthermore, an illustrative example is presented in which the
precise spatiotemporal control of one signaling pathway can
strongly influence the spatiotemporal regulation of another
signaling cascade. As regulation of any cellular processes is
achieved by an integrated network of signaling molecules, this
type of spatiotemporal cross-regulation is likely to be wide-
We thank Lonny R. Levin for the PC12 cell line and KH7 and
Ronald Schnaar for suggestions and assistance. We also thank
Viacheslav Nikolaev for providing Epac1-camps and Karal Svoboda
for providing both EKAR variants.
This work was funded by NIH grant R01 DK073368 (to J.Z.).
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