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: email@example.com.
† 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.
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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VOL. 31, 2011 SPATIOTEMPORAL REGULATION OF GF-MEDIATED PKA ACTIVITY4075